Insoluble chelating compositions

ABSTRACT

The present invention relates to insoluble compositions, which are capable of removing metal (e.g. selectively) from solution (e.g. Fe 3+  from a liquid nutrient medium so as to lower the Fe 3+  content to less than 0.1 μM); the insoluble compositions comprise: a suitable insoluble carrier and organic co-ordinating sites covalently fixed to the surface of said carrier, said co-ordinating sites being capable of chelating Fe 3+ , Th 4+  and/or UO 2   2+ .

This is a division of application Ser. No. 469,431, filed Feb. 24, 1983,U.S. Pat. No. 4,530,963, which is a continuation-in-part of applicationSer. No. 417,376, filed Sept. 13, 1982, and now abandoned.

The present invention relates to a composition useful for the removal ofmetals, in particular iron, from liquid media. The composition, can forexample, be used to lower the iron concentration of a liquid medium toless than 0.1 μM.

BACKGROUND OF THE INVENTION

Iron is an essential nutrient for all living things; a large number ofcellular enzymes and other proteins require iron in order to functionproperly. Although iron is amongst the most plentiful of metals, it isdifficult for biological systems to acquire; in aerobic environments ofsubstantially neutral pH, iron exists as its oxidized Fe³⁺ form whichreadily hydrates to highly insoluble Fe(OH)₃ polymeric forms. To ensureaccessability of iron in their environment, aerobic and facultativemicro-organisms synthesize and release into their environment highlyselective iron chelating agents called siderophores, the function ofwhich is to provide the microbes with this vital nutrient. Thesiderophores released by the microbes solubilize iron, putting it into aform readily usable by them. Thus, a free, microbial siderophore is agrowth promoting substance for those organisms which can utilise theparticular siderophore in question.

SUMMARY OF THE INVENTION

In accordance with the present invention it has been determined thatremoval of iron (e.g. Fe³⁺) from a liquid nutrient medium willsubstantially restrict the proliferation of microbes provided that theresidual iron concentration in the medium is below 0.1 μM; and thisnotwithstanding that the other required nutrients may be present inamounts sufficient for the support of microbial growth.

Thus, it is advantageous to have compositons able to remove iron from aliquid, nutrient medium since the absence or limited presence of ironwill inhibit microbial growth in such a medium. For example, thespecific removal of iron from an ophthalmic solution will inhibitmicrobial growth and spoilage of such a solution. The removal of ironfrom such a solution would obviate the addition of conventionalmicrobial growth inhibitors which can create their own problems such astoxicity, etc.

Removal of iron from liquid media prior to the present invention didpresent problems (see Neilands, J. Bacteriology 149: 880, 1982).Commercially available products (e.g. Chelex 100 sold by BioRad) have alow selectivity for iron. Additionally, other important cations (Mn²⁺,Mg²⁺) removed by such commercial products are often desirable componentsof a liquid medium. Commercial ion exchange products can also liberatesodium or potassium ions into the liquid medium being treated which maynot be desirable.

Thus, it would be advantageous to be able to remove iron from solutionwhile at the same time avoiding liberating into the solution undesirableions.

In general, it would be advantageous to be able to remove iron fromsolution when the presence of iron is undesirable, e.g. when iron isconsidered a contaminant at concentrations greater than 0.1 μM.

Free microbial siderophores cannot be used to remove iron from a liquidmedium; the natural purpose of such siderophores is to make iron solubleand available to microorganisms. The addition of free siderophores to aliquid medium would therefore enhance the growth of microorganisms whichcould utilize iron solubilized thereby. Additionally it would beextremely difficult at the very least, to recover such siderophoresloaded with iron.

Nevertheless, it would be advantageous to be able to make use ofproperties of siderophoric compounds such as microbial siderophores andother organic compounds which can provide co-ordinating groups orligands for the chelating of iron (i.e. Fe³⁺).

In accordance with the present invention, it has also been determinedthat microbial siderophores and other organic compounds possessing thesame or similar co-ordinating groups or ligands can, in addition toFe³⁺, remove Th⁴⁺ and UO₂ ²⁺ from solution. If desired, it is alsopossible to separate Th⁴⁺ from UO₂ ²⁺ if their ions are present insolution in combination.

The present invention in general relates to insoluble compositions,which are capable of removing metal (e.g. selectively) from solution(e.g. Fe³⁺ from a liquid nutrient medium so as to lower the Fe³⁺ contentto less than 0.1 μM); the insoluble compositions comprise:

(i) a suitable insoluble carrier and

(ii) organic co-ordinating sites covalently fixed to the surface of saidcarrier

said co-ordinating sites being capable of chelating Fe³⁺, Th⁴⁺ and/orUO₂ ²⁺.

In accordance with the present invention the necessary co-ordinatingsites may, for example, be provided by fixing an organic chelatingcompound such as a microbial siderophore to a suitable carrier (infra).

Thus in accordance with one aspect of the present invention, there is,in particular, provided a method for inhibiting microbial growth in aliquid nutrient medium containing Fe³⁺ by lowering the Fe³⁺ contentthereof to less than 0.1 μM characterized in that said medium iscontacted with an insoluble siderophoric composition and thereafter saidinsoluble siderophoric composition loaded with Fe³⁺ is separated fromsaid medium, said insoluble siderophoric composition comprising:

(1) one or more organic siderophoric compounds, covalently fixed to thesurface of

(2) a suitable insoluble carrier,

said organic siderophoric compounds possessing one or more co-ordinatingsites capable of chelating Fe³⁺.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

An insoluble siderophoric composition, for the purposes of this aspectof the invention, is a composition having a chelating activity withrespect to iron, and in particular a selective chelating activity.

The organic co-ordinating sites of suitable organic siderophoriccompounds may, for example, be provided by groups selected from theclass consisting of

(a) N-substituted hydroxamate groups of formula ##STR1## (b) phenolategroups of formula ##STR2## X being an atom of O or N-- (c) catecholategroups of formula ##STR3## X' being an atom of O or N-- and m being 0 or1, and (d) mixtures of two or more of the above groups; see below.

If desired, a catechol (1,2 dihydroxybenzene) compound, as definedhereinafter may be used as a siderophoric compound to providecatecholate type co-ordinating sites.

The insoluble siderophoric composition can for example be used tospecifically remove iron from liquid media such as water, juices, wine,beer, cider, chemical solutions, microbial and tissue culture media,pharmaceutical media, etc.

In accordance with another aspect of the present invention there isprovided an insoluble composition comprising a member selected from theclass consisting of

(A) an insoluble composition comprising

(1) one or more organic chelating compounds, covalently fixed to thesurface of

(2) a suitable insoluble carrier,

said organic chelating compounds possessing one or more co-ordinatingsites, said organic chelating compounds being selected from the classconsisting of microbial siderophores and

(B) an insoluble composition comprising

(1) one or more catechol compounds covalently fixed to the surface of

(2) a suitable insoluble carrier,

said catechol compounds being covalently fixed to the surface of saidcarrier at the benzene ring thereof, said catechol compounds beingselected from the group consisting of unsubstituted catechol andcatechol substituted on the benzene ring by one or two electrophilicsubstituents.

The above compositions, in accordance with this other aspect of thepresent invention can be used to remove Fe³⁺, Th⁴⁺, UO₂ ²⁺ and mixturesthereof from solution.

Thus this other aspect of the present invention also provides a methodfor removing Fe³⁺, Th⁴⁺, UO₂ ²⁺ and mixtures thereof from solutioncharacterized in that the solution is contacted with an insolublecomposition as defined above. Thereafter, the composition loaded withmetal may be separated from the treated solution. For example, the ironcontent of a liquid medium amy in this way be lowered to less than 0.1μM. Thus an insoluble composition in accordance with this aspect of thepresent invention may advantageously be used as a siderophoriccomposition to remove Fe³⁺ from liquid nutrient medium.

In particular, this aspect of the present invention also provides amethod for inhibiting microbial growth in a liquid nutrient mediumcontaining Fe³⁺, by lowering the Fe³⁺ content thereof to less than 0.1μM characterized in that said medium is contacted with an insolublecomposition as defined above and thereafter said composition loaded withFe³⁺ is separated from the medium. The iron loaded composition can, forexample be recovered by filtration.

Compositions as defined above, loaded with Fe³⁺, Th⁴⁺ or UO₂ ²⁺, maypossibly be regenerated by chemical means suitable for the removal ofthe chelated metal; the so regenerated composition can thereafter berecycled for further use.

The insoluble compositions referred to above have a very high affinityfor Th⁴⁺ and UO₂ ²⁺. They can be used to remove Th⁴⁺ and UO₂ ²⁺ fromsolution even if present in trace amounts, e.g. to obtain solutionscontaining <0.2 nM of these ions.

The present invention thus provides not only a mechanism for the removalof Fe³⁺, Th⁴⁺ and UO₂ ²⁺, from liquid media but also for thepreservation of various liquid media through the removal of irontherefrom, i.e. rendering liquid nutrient media highly resistant tomicrobial growth since any microorganism present cannot proliferate dueto the insufficient amount of iron present.

In accordance with a further aspect of the present invention there isprovided a method for treating a composition loaded with Th⁴⁺ and UO₂ ²⁺to separate Th⁴⁺ therefrom, characterized in that said composition iscontacted with an aqueous solution containing a suitable Th⁴⁺ chelatingagent and an organic acid, said organic acid being a carboxylic acid,said solution having a pH greater than 2, said composition comprising amember selected from the class consisting of

(A) an insoluble composition comprising

(1) one or more organic chelating compounds covalently fixed to thesurface of

(2) a suitable insoluble carrier,

said organic chelating compounds possessing one or more co-ordinatingsites, said organic chelating compounds being selected from the classconsisting of microbial siderophores, and

(B) an insoluble composition comprising

(1) one or more catechol compounds covalently fixed to the surface of

(2) a suitable insoluble carrier,

said catechol compounds being covalently fixed to the surface of saidcarrier at the benzene ring thereof, said catechol compounds beingselected from the group consisting of unsubstituted catechol andcatechol substituted on the benzene ring by one or two electrophilicsubstituents.

In accordance with this further aspect of the present invention, it ispossible to separate Th⁴⁺ and UO₂ ²⁺ which are present in a solution.Thus, for example, an aqueous solution containing Th⁴⁺ and UO₂ ²⁺ andhaving a pH of about 7 can be contacted with an insoluble composition asdefined above, (preferably a composition incorporating a catecholcompound fixed to a silica based carrier), to give rise to a compositionloaded with Th⁴⁺ and UO₂ ²⁺. This loaded composition can then berecovered and treated, as outlined above to separate the Th⁴⁺ from thecomposition, to provide a treated composition loaded with UO₂ ²⁺ buthaving a substantially reduced Th⁴⁺ content. The treated composition canthen be contacted with, for example, an aqueous acidic solution torecover a concentrated UO₂ ²⁺ solution; for example a compositionincorporating a catechol compound fixed to a silica based carrier can betreated with an aqueous mineral acid (e.g. HCl) having a pH≅0.8-1.5 torecover a concentrated UO₂ ²⁺ solution.

The Th⁴⁺ and UO₂ ²⁺ solution can for example, be provided by treating ina known manner, radioactive materials from atomic reactors. For example,Th can be converted to U by treating solid ²³² Th with neutrons whichconvert ²³² Th to ²³³ Th. The ²³³ Th decays to a fissionable type ofuranium i.e. ²³³ U. The mixture of these metals can then be solubilizedwith a suitable agent such as HNO₃. The obtained solution, onceneutralized to a pH >4 can then be treated for the separation of themetals as outlined above, i.e. to produced a concentrated solution ofthe fissionable type of uranium.

Organic chelating compounds, e.g. sidephoric compounds, useful inaccordance with the present invention may also form complexes withcertain other transition, rare earths and actinide metals due to thestructural (atomic) similarities with iron; however, the complexes areformed at lower affinities than, for example UO₂ ²⁺ or iron. Althoughthe compositions of the present invention can possibly be used for theremoval of these other metals from liquid media, the followingdiscussion will be directed to the removal of Fe³⁺, Th⁴⁺ and UO₂ ²⁺ fromliquid media.

In accordance with the present invention, a general process for thepreparation of an insoluble composition as defined above can becharacterized in that organic co-ordinating sites capable of chelatingmetal are covalently fixed to the surface of a suitable carrier. Anysuitable means of covalently fixing organic co-ordinating sites to acarrier can be used provided that the composition obtained has thenecessary chelating activity.

If it is desired to produce a siderophoric composition comprising one ormore organic siderophoric compounds fixed to a suitable carrier then theprocess of its preparation may be characterized in that a suitablecarrier is reacted with one or more organic siderophoric compoundspossessing co-ordinating sites capable of chelating Fe³⁺ so as tocovalently bond said siderophoric compounds to the surface of saidcarrier, while maintaining the Fe³⁺ chelating activity of saidsiderophoric compounds.

The siderophoric compounds as indicated above can be microbialsiderophores. In this particular case, the process of preparation can becharacterized in that a suitable carrier is reacted with one or moremicrobial siderophores so as to bond said microbial siderophores to thesurface of said carrier, said carrier and said microbial siderophorespossessing functional groups reactive one with the other so as tocovalently bond said microbial siderophores to said carrier whilemaintaining the Fe³⁺ chelating activity of said microbial siderophores.

Turning now to the chelating compounds, sufficient metal coordinationsites to chelate the metal ions (i.e. Fe³⁺, Th⁴⁺ and UO₂ ²⁺) may beprovided by a single organic chelating (e.g. siderophoric) compound oralternatively by two or more such compounds; the number of compoundsparticipating in the chelation of the metal ions being dependent uponthe number of coordinating sites which are available from a particularcompound fixed to a carrier.

The organic chelating compound used can as indicated above be amicrobial siderophore. A microbial siderophore may have a molecularweight of less than 2500 Daltons, e.g. a molecular weight in the rangeof 500 to 2500 Daltons. A microbial siderophore useful in accordancewith the present invention can also possess one or more types of metalcoordinating sites within its structure. The sites can be provided bygroups selected from the class of groups referred to earlier, e.g.N-substituted hydroxamate groups, catecholate groups etc. Siderophorespossessing these groups display high selectivity and very highaffinities for Fe³⁺, Th⁴⁺ and UO₂ ²⁺.

A representative list of microorganisms and their siderophores is givenin following table 1:

                  TABLE 1                                                         ______________________________________                                                        COMMON NAMES                                                                  OF SIDEROPHORE                                                ORGANISM NAME   OBTAINED THEREFROM                                            ______________________________________                                        Prokaryotes                                                                   Enteric species Enterobactin (enterochelin),                                                  Aerobactin                                                    Agrobacterium tumefaciens                                                                     Agrobactin                                                    Pseudomonas species                                                                           Pyochelin, Pyoverdine,                                                        Pseudobactins, Ferribactin                                    Bacillus megaterium                                                                           Schizokinen                                                   Anaboena species                                                                              Schizokinen                                                   Arthrobacter species                                                                          Arthrobactin                                                  Azotobacter vinelandii                                                                        α,ε-bis-2,3,-dihydroxybenzoyllysine             Actinomyces species                                                                           Ferrioxamines                                                 Mycobacterium species                                                                         Mycobactins                                                   Eukaryotes (Fungi)                                                                            Ferrichromes, Copragen                                        Pencillium species,                                                           Aspergillus species,                                                          Neurospora, Ustillago                                                         Rhodotorula species                                                                           Rhodotorulic acids                                            Ectomycorrhizal species                                                                       Hydroxamate type                                              ______________________________________                                    

The basic chemical structure, trivial names and possible sources of sometypes of microbial siderophores are listed below; the term microbialsiderophore of course includes any suitable functional derivatives,analogs or enantioforms of these molecules: ##STR4## wherein:

R=H or --COCH₃ ; R'=CH₃ -- or HOOC--(CH₂)₂ --; n=4 or 5.

For ferrioxamine B, R=H and R'=CH₃ --. The mesylate salt ofdeferrioxamine is marketed by Ciba-Geigy as Desferal (U.S. Pat. Nos.1964 3,118,823 and 3,153,621; Can. Pat. Nos. 1962 648981 and 715051.##STR5## wherein:

R', R", R'"=H

R=--CH₃

Ferrichrome

(prototype) ##STR6## wherein:

R₁ =methyl, ethyl or alkyl or alkenyl of 11 to 20 carbon atoms

R₂ =H or methyl

R₃ =H or methyl

R₄ =methyl, ethyl, or alkyl of 15 to 18 carbon atoms

R₅ =H or methyl ##STR7## wherein:

R=H,OH ##STR8##

A detailed description of the above siderophores is given by Neilands(Annu. Rev. Biochem. 50: 715-731, 1981), and their coordinationchemistry has been reviewed by Raymond and Carrano (Accounts of ChemicalResearch, Vol. 12, No. 5 (1979), at pages 183-190).

Microbial siderophores can be extracted for example from spent microbialculture media with organic solvents. Examples of such methods are givenin U.S. Pat. Nos. 3,118,823 and 3,153,621 as well as Canadian Pat. Nos.648,981 and 715,051. For example, siderophores possessing hydroxamateligands may be obtained in this fashion. Hydroxamate microbialsiderophores are distributed widely throughout the prokaryotic andeukaryotic microbial world, but to date, only bacteria are known whichproduce typical mono- and dihydroxybenzoic acid-bearing siderophores.

Some microbial siderophores, their analogs and/or their enantioformshave been chemically synthesized in the laboratory:

(i) enterobactin, its enantioform and carboxylic, methyl, and aromaticanalogs;

(ii) N¹, N⁸ -bis-2,3-dihydroxybenzoylspermidine;

(iii) ferrichrome and enantio-ferrichrome.

Other processes for the preparation of various siderophores aredescribed in Canadian Pat. Nos. 742,670, 746,873, 773,540 and 775,539.

A discussion of the preparation of siderophores by denovo synthesis canbe found in Neilands Review 1981 and Neilands et al J. Biol. Chem. 256;3831-3238, 1981.

The ferrioxamine B is sold commercially under the designation Desferalwhich is a trademark of Ciba-Geigy.

The microbial siderophores, ferrioxamine and enterobactin, referred toabove are prototypical natural microbial siderophores and eachrepresents the general structure and properties of hydroxamate andcatecholate-bearing siderophores respectively. These particularsiderophores will be referred to below (e.g. in the examples). For thepurposes of this specification the expression des as it appears beforeferrioxamine etc is to be understood to refer to ferrioxamine etcwherein co-ordinating sites are unoccupied e.g. they are not ironloaded.

Turning now to carriers suitable in accordance with the presentinvention, they must of course be insoluble in the liquid medium ofintended use; for example, the carrier can be water insoluble.Desirably, the carrier is also inert in the liquid medium of intendeduse. The carriers can be in particulate or solid form.

The carrier can be an organic or inorganic compound. For example, thecarrier may be a natural or modified natural polymer (e.g. lignin, agar,alignate, glucan, cellulose, dextran, cellulose acetate, humic acid,etc.) a synthetic organic polymer (e.g. a polyamide, a polyamine, apolyacrylamide, a polyester, a polyurethane, a polyethylene, apolystyrene, a polypropylene, a polycarbonate, a silicone, nylon, latex,a polyfluroolefin, etc.) or an inorganic material (a ceramic, a glass,carbon, etc.).

As indicated above the present invention provides a (siderophoric)composition comprising one or more microbial siderophores which arecovalently immobilized or fixed on a suitable insoluble carrier in sucha way that the microbial siderophores retain their high chelatingaffinity for metal ions, i.e. iron.

A number of known processes are suitable for the binding of microbialsiderophores to carriers so as to preserve the iron chelating orcomplexing properties thereof. For example, the commonly used methodsfor covalently binding enzymes to insoluble carriers can be adapted forthe immobilization of microbial siderophores. See, for example <<Methodsof Enzymology>>, XXXIV B:30 (Jakoby W. B. Ed.) Academic Press, New York(1974).

Carriers which are suitable for the process of preparing siderophoriccompositions using microbial siderophores are those which have activesurfaces; the active surfaces have functional groups which can bond to acompatible functional group of the chosen siderophoric compound. Thefunctional group can, for example, be selected from the class consistingof ##STR9## --(CH₂)_(n) --NH₂, n being 0, 1, 2, 3, etc. ##STR10## Xbeing a halogen atom, for example, Br, ##STR11## X being, as definedabove, ##STR12## However, any functional group can be used which willreact with a functional group on the microbial siderophore in questionto bind it to the carrier, the microbial siderophore retaining its ironchelating capacity.

It is possible to put some distance between a microbial siderophore andthe surface of the carrier, e.g. in order to limit the effect on themicrobial siderophore of a surface characteristic of the carrier. Forexample, teflon may be used as a carrier. However, teflon has a highlyhydrophobic surface which is non-wetting. Therefore, it is desirable toput some distance between the surface of the teflon and the microbialsiderophore to allow the siderophore to extend well into an aqueousliquid medium.

A spacer compound may be used to provide a spacer group to space apart acarrier and a siderophore.

A suitable spacer compound is bifunctional; i.e. it has a functionalgroup which can react with a functional group of the carrier to bind itthereto; and it has also a second functional group which can react witha compatible functional group on the chosen microbial siderophore tobind it thereto: see the above groups. The spacer group mayalternatively have a second functional group which while not reactivewith a compatible functional group on the siderophore, may beconvertible into such a group.

A spacer compound can, for example, in addition to the above referred tofunctional groups, include a hydrocarbon chain, the length of which ischosen in accordance with the distance which it is desired to placebetween the carrier and the siderophore. The spacer compound used may be1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride salt orglutaraldehyde. However any compound can be used which will space themicrobial siderophore from the carrier, the necessary or desireddistance provided of course that it is bifunctional.

The spacer compound may be bound, to a carrier by making use ofconventional reactions involving the formation of ester groups, amidegroups, amino groups, diazo groups, ether groups, sulphonamide groups,amidino groups; the reaction may be a carbon-carbon condensation.

Thus a carrier suitable for the process of the present invention may berepresented generally by the formula ##STR13## wherein n is an integer,"back" is a carrier backbone, "R" is a single bond or a suitable spacergroup and "ƒcn" is a functional group as defined above. For example"ƒcn" may be a carboxyl group and R may be a group such as ##STR14##

A useful carrier may need to have its surface treated in order toprovide the surface with a suitable functional group which can bond to amicrobial siderophore.

Thus silica (e.g. in the form of a silica gel) having surface hydroxylgroups can for example, be pretreated with a suitable ω-amino(C₂ to C₁₀alkyl) tri (C₁ to C₅ alkoxy) silane to provide an active surfacecomprising amino groups. The silane can, for example, beγ-aminopropyltriethoxysilane. See, for example, the following patentswherein silica is treated with a silane: Canadian Pat. Nos. 1,102,347,1,103,035 and 1,102,346; U.S. Pat. Nos. 4,203,952, 3,886,080, 3,904,373,3,519,538, 3,652,761, 4,230,803, and 4,290,892.

Nylon, for example, is a carrier which requires a pretreatment toprovide it with suitable functional groups. Since the nylon contains theamide group, its surface may be subjected to partial hydrolysis usingknown techniques to give free amino and carboxyl functional groups. Theaqueous method may proceed as below: ##STR15##

Alternatively, nylon can be reacted in a non-aqueous medium with, forexample, thionylchloride to give rise to the functional group ##STR16##If desired, an appropriate spacer group can be readily attached through,for example, the use of ethylene diamine or another functionallyequivalent species such as ##STR17## followed by a reduction of --NO₂ to--NH₂ suitable for generation of diazonium salts which are then suitablefor coupling to a microbial siderophore, e.g. enterobactin. Succinicanhydride can thereafter be used as a further extension of the spacergroup; i.e. to form an amide linkage.

Teflon is another useful carrier which must be pretreated in order toprovide it with a suitable functional group which can bond to amicrobial siderophore.

Teflon, a tradename for polytetrafluoroethylene from DuPont, is highlyinert and is not readily attacked by acids and bases. No easydisplacement of the fluorine atomes is known. Fluorine atom can,however, be displaced by ion-radicals such as sodium or potassiumnapthalene of formula: ##STR18##

On reaction between teflon and such sodium or potassium naphthalene, asodio or potassio species of teflon is formed of formula: ##STR19##These organo metallic species of teflon are highly reactive towards manyorganic functional groups and their general behaviour is similar to thewell known Grignard reagents. Thus, they can be reacted with a dimethylcarbonate to give rise to an alkoxy carbonyl substitutedpolytetrafluoroethylene. This substituted ethylene can subsequently besubjected to hydrolysis to provide a polytetrafluoroethylene withcarboxyl substituents. The carboxylated teflon thus generated, can thenbe used for direct coupling to microbial siderophores (or chelators)such as "Desferal". As indicated above, it may be desired to space thesiderophore from the surface of the teflon. If so, ethylenediamine andsimilar compounds can be readily attached through the carboxyl group bystandard procedures. Since the teflon's backbone is very inert to manyorganic and inorganic reagents, very vigorous reaction conditions can beemployed in further derivatization using the carboxylic functionalgroup. See, for example, "Methods in Enzymology" Supra.

As indicated above, the microbial siderophore must also possesscompatible functional groups which will react with those of the carrierswithout interfering with the chelating activity thereof. For example,suitable functional group in the siderophore enterobactin is the 2,3-dihydroxy benzoic group which is susceptible to diazonium couplingunder neutral to almost neutral conditions. The acylamine required inthe generation of diazonium salts can be prepared fromaminopropylsilylated glass.

Examples of suitable functional groups on the microbial siderophores arethe amino group, the carboxyl group, the phenolate group and thecathecolate group, etc.

The previous comments relating to carriers, spacer groups etc formicrobial siderophores apply to the use of other organic chelating (e.g.siderophoric) compounds, for example a catechol compound. If a catecholcompound is used as the chelating compound, it may be bonded to acarrier by diazo coupling or through reaction with a functional group onthe carrier such as ##STR20## In these latter cases the functional groupon the carrier is directly reactive with the benzene ring of thecatechol.

The preparation of a composition comprising unsubstituted catecholsuitably fixed to a silica gel carrier can be graphically described asfollows: ##STR21##

The above catechol compositions will hereinafter be referred to as##STR22##

The above catechol compositions, e.g. Sili ##STR23## if stored for aprolonged period of time loaded with Fe³⁺, undergo a chemical changewhereby the composition gradually looses chelating activity. It isbelieved that this loss of activity is due to the reciprocal oxidationof the catechol and reduction of Fe³⁺ (i.e. Fe³⁺ →Fe²⁺). The activity ofthe composition may be recovered by treating the composition with asuitable reducing agent such as will be discussed further on.

It has been determined that catechol compounds selected from the groupconsisting of catechol substituted on the benzene ring by one or moreelectrophilic substituents (i.e. the ring is mono or di-substituted) notonly have an activity similar to that of unsubstituted catechol but havethe additional advantage that they are better able to maintain theiractivity notwithstanding prolonged periods of iron loading. Accordinglythese compounds can be used in circumstances where it is desired toavoid a reduction treatment. The substituents can be added to thebenzene ring once catechol is fixed to the desired carrier. Thesubstituent can be chosen for example from the class consisting ofHalogen atoms (e.g. Cl and Br), NO, NO₂, COOH and ##STR24## The ring canbe mono or di halo substituted or mono substituted with NO or NO₂. Thesubstituted catechol residues may thus have the following formulae##STR25## wherein R is a substituent as defined above, n may be 1 or 2.

When using a composition in accordance with the present invention, theconditions of use should of course be such as to avoid the break-down ordecomposition of the composition; i.e. conditions such as <<pH,temperature, pressure, etc.>> should be chosen so as to avoid thebreak-down of the composition.

As indicated above, an insoluble (siderophoric) composition, inaccordance with the present invention, can be used to remove iron from aliquid medium. In use, the (siderophoric) composition is intermixed witha desired liquid medium for a suitable time, which will of course dependupon the amount of (siderophoric) composition used, the initial ironconcentration, the desired final iron concentration, etc. The Fe³⁺ inthe medium combines with the (siderophoric) composition and can thus bephysically separated from the medium. The affinity of (siderophoric)compounds for iron can be so great that even small amounts of iron canbe removed from a liquid medium. The final concentration of iron in atreated nutrient medium can for example be far below that required tosupport microbial growth.

In drawings which illustrate the present invention

FIG. 1 is a graph illustrating the inhibition of microbial growth due tothe removal of iron from a liquid medium,

FIG. 2 illustrates regeneration of a siderophoric composition by pHmanipulation; and

FIG. 3 illustrates regeneration of a siderophoric composition by areducing agent.

FIG. 1 as indicated above is a graph illustrative of the inhibition ofmicrobial growth in even the most nutritional solutions (e.g.bacteriological broth media) on removal of iron therefrom.

In particular, FIG. 1 illustrates the inability of the bacteriumNeisseria meningitidis to grow in a complex highly nutritional medium(neisseria defined medium--NDM) from which only iron has been extractedwith ferrioxamine immobilized on agarose, the agarose having previouslybeen activated by cyanogen bromide for coupling to ferrioxamine. Thus,one gram of the above siderophoric composition was contacted with 200 mlNDM at 22° C. for a time period of 20 min. before recovering thesiderophoric composition. Prior to treatment, the NDM contained about3.6 μM of iron; after treatment, it contained less than 0.1 μM iron. Thetreated medium was then divided into two portions and FeCl₃ was added toone of them. A control consisting of untreated NDM and the two portionswere then inoculated with microbes and maintained at a pH of 7.4 and atemperature of 37° C. As can be seen in FIG. 1, unhibited growth occursin the control (0). However, in the treated medium, (), cells are unableto undergo anymore than one or two divisions due to the absence of thevital nutrient iron. On the other hand if exogenous iron is added backto the treated medium full growth is again realized ().

Liquid media to be treated to remove Fe³⁺ can have, for example, a pH inthe range of 4.5 to 9. During the contact with the siderophoriccomposition, the temperature of the mixture can for example range from1° C. to 50° C. and the contact can occur under atmospheric pressure.Examples of different media which can be treated with the compositionare listed in Table 2 which follows:

                  TABLE 2                                                         ______________________________________                                        Classes of liquid media                                                                       Specific example thereof                                      ______________________________________                                        Liquid foods    fruit and vegetable juices, clear                                             meat broth (e.g. consomme), culture                                           media for microbial, plant and                                                animal cells                                                  Beverages       wine, beer, natural and synthetic                                             juices, cider, drinking water                                 Pharmaceutical  buffer solutions for lavage (e.g.                                             ophthalmic solution, peritoneal                                               lavage), water used in the manu-                                              facture or various solutions and                                              preparations, antibiotic solutions,                           Cosmetics (liquid)                                                                            those susceptible to microbial                                                degradation, contamination, or                                                spoilage                                                      Industrial water and                                                                          cooling tower, process and waste                              waste water     water                                                         Natural water   removal of actinides (e.g. Th.sup.4+, UO.sub.2.sup.2+)                        and chromium                                                  ______________________________________                                    

A siderophoric composition can, as indicated above, for example, be usedto remove iron from microbial fermentation cultures to stop furthergrowth of microbes in the fermenter. Thus, a siderophoric composition inaccordance with the present invention may be used to treat wine in orderto inhibit microbial growth therein.

The composition of the present invention may also be used to remove ironfrom cosmetic solutions to prevent contamination by the growth ofmicrobes. Components for cosmetic solutions are often obtained fromnatural sources and are susceptible to microbial degradation.

A siderophoric composition of the present invention may also be used forthe removal of iron from drinking water, pharmaceutical and biologicalsolutions, and industrial water.

Although the microbial siderophores are selective for iron, they canalso bind metals that are classified as actinides e.g. uranium. Thus,the present invention additionally provides means for removing suchhazardous metals as plutonium from contaminated water; and a rapid meansto collect (concentrate) the radioactive heavy metals (e.g. plutonium)to determine the concentration thereof in standard water volumes. Suchmetals are selectively removed from water due to their structuralsimilarity (i.e. atomic) to iron.

As indicated previously, compositions in accordance with the presentinvention, may possibly be regenerated for further use by the removal ofthe metal therefrom by suitable chemical means. In this way, thecomposition can be economically used since it can be recycled forrepeated use.

The regeneration, for example, of a (siderophoric) composition loadedwith iron, may be carried out either through the manipulation of the pHof a medium surrounding the iron-loaded (siderophoric) compositionand/or by treating the iron-loaded composition with a suitable reducingagent. In either case, appropriate conditions should be chosen whichwill not decompose the composition or destroy the iron binding capacitythereof.

If regeneration is affected by manipulation of the pH, the pH must bebrought to or beyond a point at which the iron is released.

In general, when making use of a microbial siderophore, a pH of 1 orlower should be avoided; the use of mineral acids should also beavoided. The pH can be manipulated through the use of organic acids (forexample, acetic acid, succinic acid, citric acid, isocitric acid,ketomalonic acid, malic acid, oxalic acid or pyruvic acid).

If a catechol compound is used a mineral acid may be used to manipulatethe pH.

FIG. 2 illustrates the regeneration of a (siderophoric) composition bythe manipulation of pH. The designation () represents enterobactinimmobilized on a polyacrylamide carrier whereas the designation (O)represents desferrioxamine immobilized to the same type of carrier. ThepH was lowered in the presence of 15 mM citrate and 0.05M tris-sodiumacetate. The lowering of the pH was accomplished by an addition ofappropriate amounts of acetic acid.

Alternatively, an iron loaded composition may be treated with a suitablereducing agent to release the iron. In accordance with the presentinvention, it is possible to use suitable dithionites or ascorbates asthe reducing agents, e.g. sodium or potassium dithionite and sodium orpotassium ascorbate. The dithionites can be used for the reduction of(siderophoric) compositions which include hydroxamate ligands whereasthe ascorbates can be used for the reduction of siderophoriccompositions containing phenolate/catecholate group ligands. Otheruseful reducing agents include hydroxylamine and hydroquinone.

The compositions wherein the organic chelating compound includescatecholate ligands (e.g. siderophoric compositions consisting ofenterobactin fixed to glass) and especially the compositions containingdiazo linkages must be subjected to mild reduction conditions such asprovided using ascorbic acid. The compositions which include hydroxamategroups (Desferal), can be reduced with 1.0 molar sodium dithionite.

Other reducing agents may possibly be used to regenerate a composition;however, the reducing agent used must be chosen on the basis that itwill not destroy the integrity or metal-binding (e.g. iron-binding)capacity of the composition. Sodium dithionite (Na₂ S₂ O₄),hydroxylamine (including its acid addition salts) and hydroquinone areas indicated above examples of useful reducing agents. In particular thereducing agent can be hydroxyl amine chloride.

The regeneration of a (siderophoric) composition may take place in thepresence of a suitable organic acid that will complex with the iron thatis released. Suitable acids are di or tricarboxylic organic acids thatwill chelate the liberated iron ions.

FIG. 3 illustrates the repeated regeneration of (siderophoric)compositions consisting of enterobactin ( ) desferrioxamine ( ) bound topolyacrylamide carriers, the reducing agent consisting of sodiumascorbate. The regeneration solution had a pH of 7 in the presence of 15mM sodium citrate and 0.05M sodium acetate.

Compositions in accordance with the present invention which are loadedwith Th⁴⁺ and/or UO₂ ²⁺ may possibly be regenerated in the same manneras for iron loaded compositions. When regenerating a composition loadedwith UO₂ ²⁺ by manipulation of pH a relatively low pH (e.g. pH≅0.8) maybe needed to remove this ion; accordingly it may be necessary to resortto the use of a mineral acid to recover the ion e.g. HCl. Less severecondition may be used to recover the Th⁴⁺ e.g. by using a wash solutionhaving a higher pH than that necessary for the removal of UO₂ ²⁺.

Since the Th⁴⁺ may be recovered at a higher pH than the UO₂ ²⁺ it ispossible to manipulate the pH of the solution to be treated such that acomposition in accordance with the present invention will preferentiallytake up the UO₂ ²⁺ thereby separating UO₂ ²⁺ from Th⁴⁺.

Alternatively as indicated above, an insoluble composition in accordancewith the present invention may be used to take up both UO₂ ²⁺ and Th⁴⁺.The so obtained composition may then be treated with an aqueous solutioncontaining a suitable Th⁴⁺ chelating agent and an organic acid, the pHof the solution being greater than 2.

Preferably the pH of the aqueous solution should be about 4 to about 6.

Some of the above referred to microbial siderophores in free form (i.e.not fixed to a carrier) may possibly be used to chelate Th⁴⁺ for theabove method. Alternatively the ion chelating agents disclosed inArchibald, F. A. and I. W. DeVoe 1980, Iron acquisition by Neisseriameningitidies in vitro Infect. Immun 27:322-334, are suitable forchelating Th⁴⁺.

The chelating agent and the organic acid may both be a tricarboxylicacid; they may be the same. It is possible to provide the necessarytricarboxylic acid by adding a mineral acid (e.g. HCl) to an aqueoussolution containing an alkali metal salt of a suitable tricarboxylicacid e.g. Na₃ citrate.

Suitable Th⁴⁺ chelating agents may be selected from among organic acidshaving the following general structures ##STR26## wherein R' and each Rare independently selected from the group consisting of hydrogen andsuitable organic residues; R' may also alternatively be OH or alkoxy (C₁to C₁₀); p and k are the same or different and are 0 or an integer; R',R, p and k are selected such that the chelating activity of the threeCOOH groups is not interfered with. R can for example be selected fromalkyl (e.g. C₁ to C₁₀); Halogen (e.g. Cl, Br); p and k may possibly be 1to 10.

Preferably each R is H and p and k are 0.

Suitable chelating agents are citric acid, isocitric acid, cis-aconiticacid and oxalosuccinic acid. The alkali metal salts of these acids couldof course be used in conjunction with a suitable source of protons (i.e.H⁺) e.g. a mineral acid such as HCl.

The organic acids may be selected from the same group of acids asoutlined above for the Th⁴⁺ chelating agents. However, other organicacids may be used such as the mono and di carboxylic acids (e.g. aceticacid, malonic acid etc.).

A pH for the aqueous solution of greater than 2 is necessary in order toinhibit the simultaneous removal of UO₂ ²⁺ from the composition i.e. ata pH lower than 2 UO₂ ²⁺ can be recovered from the composition. Thusonce Th⁴⁺ is separated from the composition it is possible to recover aconcentrated solution containing UO₂ ²⁺ by subsequently treating thecomposition with an aqueous solution having a low pH. As indicated abovesince it may be necessary to use a mineral acid in order to recover UO₂²⁺ the composition in such case can be based on a catechol compound; thecatechol compound is preferably fixed to a silica gel if the UO₂ ²⁺ isto be recovered and the composition reused.

The insoluble compositions in accordance with the present invention thusprovides for the advantageous removal of metals (e.g. iron) from liquidmedia. Such media remain essentially unchanged except for the absence ofmetal. The liquid media referred to herein may be aqueous, organic ormixtures thereof.

Reference will now be made to a number of examples which deal withembodiments of the present invention.

EXAMPLE 1 Activation Of Silica Gel (glass)

The activation methods were analogous to those as described by H. Weetal& A. M. Filbert, Methods of Enzymology XXXIV B:59-72 1974.

(a) Pretreatment

100 grams of silica gel designated Sigma S-4133 (sold by Sigma ChemicalCompany) of 100 to 200 mesh (70 to 140 microns) chromatographic gradeand of pore diameter of about 25 angstrom was suspended in a mixture of50 ml of 70% HNO₃ and 300 ml of distilled water. The suspension wasrefluxed with mixing for about 1 hour.

The gel was allowed to settle and the liquid layer drawn off. The gelwas then washed repeatedly with distilled water until the wash water wasabout neutral pH.

(b) Amine activated silica gel

The above pretreated silica gel (wet) was used for the followingamination without drying. A 10% solution of γ-aminopropyltriethoxysilanein distilled water (500 ml) was added to the above obtained silica gel.The pH was then adjusted to 3.45 with 6N hydrochloric acid. Thesuspension was then maintained under stirring at a temperature of 75° C.for about 3 hours. The gel was then filtered and washed with 500 ml ofdistilled water and dried in an oven at 100° to 110° C.

The above amination can be described graphically as follows: ##STR27##

The above obtained amineactivated silica gel will hereinafter bereferred to as glass --NH₂

(c) Aminoarylcarbonyl activated silica gel

50 grams of glass --NH₂ was suspended in 300 ml of ethanol freechloroform. 2.5 grams p-nitrobenzoyl chloride [Eastman Kodak] wassubsequently admixed therewith. Thereafter, 30 ml of dry triethylaminewas added and the resultant mixture was refluxed for 20 hours. The beadswere allowed to settle and the liquid phase drawn off. The beads werethen wash repeatedly with chloroform then repeatedly with ethanol andfinally with distilled water with ethanol. The wet water-washed gel wassubjected to a treatment for the reduction of the nitroaryl group to anaminoaryl group by adding the gel to a solution of 50 gm of sodiumdithionite in 250 ml. of distilled water. The whole suspensionthereafter being refluxed with mixing for 45 min. The suspension wasthen filtered while still hot and washed repeatedly with dilutehydrochloric acid and then washed with distilled water. After thoroughwashing with distilled water, the gel was dried in an oven at 70° to 80°C.; this type of activated carrier can be used for subsequentdiazotization and coupling.

The above reaction can be represented graphically as follows: ##STR28##

(d) Carboxyl activated silica gel

50 gm of glass --NH₂ (see above), were suspended in 250 ml of water coldin an ice bath. 50 ml of 1N sodium hydroxide was added followed by 15 gmof solid succinic anhydride. After mixing for 2 hours, the pH wasadjusted with the addition of 1N sodium hydroxide to a pH of 5-6. Thisadjudtment of pH with sodium hydroxide was repeated hourly threeadditional times and the mixture was left overnight. The suspension wasthereafter filtered and washed thoroughly with distilled water and driedat 100° to 120° C.

The above reaction can be represented graphically as follows: ##STR29##

(e) Aldehyde activated silica gel

20 gm of glass --NH₂ (see above) were suspended in 50 ml of 0.1M sodiumphosphate at a pH of 7 followed by the addition of 10 ml purified 8%glutaraldehyde. The mixture was subjected to vacuum and mixedoccasionally. The reaction was allowed to proceed for 3 hours and themixture was thereafter filtered, washed thoroughly with distilled waterand dried under vacuum.

The reaction outlined above can be represented graphically as follows:##STR30##

EXAMPLE 2 Activation Of Polyacrylamide (Azide Coupling)

60 ml of ethylene diamine activated polyacrylamide gel (per Inman,Methods in Enzymology XXXIV B:35 1974) was diluted with water to make upto 100 ml volume. About 1.2 g of p-nitrobenzoyl azide was dissolved in100 ml of tetrahydrofuran and the obtained solution was subjected tofiltration. The obtained filtrate was added immediately to the aqueousgel suspension referred to above. 1.5 ml of triethylamine was then addedto the suspension. The gel mixture was then stirred gently for 30 min.An additional portion of 1.2 grams of p-nitrobenzoyl azide in 50 ml oftetrahydrofuran was added followed by another 1 ml of triethylamine.Gentle stirring was continued for an additional hour. The obtained gelwas filtered and washed thoroughly with 1:1 tetrahydrofuran:0.2M sodiumchloride and then resuspended in 0.2M sodium chloride. 3 ml of aceticanhydride was added to the suspension which was then mixed for 1 hour,the gel being thereafter washed with 0.1M NaCl.

The obtained gel can be used directly for diazo coupling. The reactioncan be represented graphically as follows: ##STR31## wherein:B=polyacrylamide backbone (ie carrier)

EXAMPLE 3 Activation Of Agarose With Cyanogen Bromide

Agarose swollen in water was mixed with an equal volume of water.Finally divided CNBR (50-300 mg per ml of agarose) was added at once tothe stirred suspension. The pH of the suspension was immediatelyadjusted and maintained at pH 11 with sodium hydroxide. The temperatureof the suspension was maintained at 20° C. and the reaction allowed toproceed for about 30 min. Thereafter, the gel was then washed rapidlywith a large amount of ice cold water followed by washing with anappropriate buffer. The obtained activated gel should be used as soon aspossible.

The chemical reactions involved in the agarose activation can beindicated graphically as follows: ##STR32## Cyanogen bromide, byanalogous procedures, can be used to activate other polysaccharides; forexample, alginate, glucans, cellulose, agar, dextrans, etc.

EXAMPLE 4 Immobilization Of Ferrioxamine Or Enterobactin

1 mM HCl-washed, water swollen CNBR activated carrier, obtained inaccordance with Example 3, was mixed with ferrioxamine or enterobactin(20 mg/ml) in an NaHCO₃ buffer (0.1M, pH 8.3) containing 0.5M NaCl. Themixture was agitated overnight at 4° C. The gel was then washed with0.1M acetate buffer pH 4.0 containing 0.5M NaCl to remove excessuncoupled ferrioxamine or enterobactin.

EXAMPLE 5 Immobilization Of Desferal To Polyacrylamide Gel

(a) Biogel P-150 polyacrylamide from BioRad was linked throughethylenediamine and then succinic anhydride following publishedprocedures as described by Inman (John K. Inman, Covalent Linkage ofFunctional Groups, Ligands, and Proteins to polyacrylamide beads in"Methods of Enzymology", XXXIV B, 30 (Jakob, W. B. ed.) Academic Press,New York (1974) and Biochemistry 8:4074 (1969); (see examples 16 and 17)infra. Unreacted free amino groups in ethylene-diamine were blocked byreaction of acetic anhydride at the end of the reaction period. Test forpresence of any free amino group was through the TNBS (trinitrobenzenesulfonic acid) test. The addition of acetic anhydride was repeated twiceuntil TNBS test were negative.

This activated, extended polyacrylamide was immediately used forcoupling.

(b) Coupling of Desferal to activated polyacrylamide gel

700 mg Desferal was dissolved in 5 ml of deionized water and followed byaddition of 163 mg ferric chloride. A deep red solution was formed. 20ml activated gel, obtained above, was washed twice with ethanol bycentrifigation and decantation. The total solid shrank to a very smallvolume after the second volume of ethanol (20 ml) was added. The 5 ml offerric complexed Desferal was added at 20° C. to this shrunken gel andthe mixture was mixed by swirling. The gel gradually swelled to give asolid mass. 5 ml of H₂ O was added to help disperse the gel and the pHof the suspension was adjusted to 4.2 with NaOH (1N). 200 mg EDC(1-ethyl-3-(3-dimethylamino propyl)carbodiimide), 200 mg was added inone portion and the pH of the suspension monitored and kept at 4.3 to4.6 by addition of HCl (1N), for 3 hrs. A further portion of 200 mg EDCwas added and the mixture, allowed to stand overnight at roomtemperature. The gel was filtered on a sintered flass funnel and washedwith 0.2M NaCl.

The reaction can be graphically represented as follows: ##STR33##wherein: PA=polyacrylamide backbone or carrier and

De--=desferal.

EXAMPLE 6 Immobilization Of Ferrioxamine Or Enterobactin ToPolyacrylamide Carrier

A swollen polyacrylamide gel acyl azide derivative freshly prepared asin accordance with example 2 was suspended in a solution containing thefollowing: 0.1M CaCl₂, 0.001N HCl acid, ferrioxamine or enterobactin at0.3 mg/ml (pH 4.0). The pH was immediately adjusted to 9.0 and themixture was stirred for 60 min. at 0° C. In the case of enterobactin,the solutions are 50% ethanol. The coupled gel was washed with largevolumes of 0.05M Tris-acetate-0.15M citrate, pH 7.0.

EXAMPLE 7 Immobilization Of Desferal To Silica Gel (Glass Beads)

About 700 mg (or approximately one mMole) of Desferal was dissolved in25 ml of water followed by the addition of 170 mg of ferric chloride.The pH of the solution was then adjusted to 4.3 with one normalhydrochloric acid and to this was added 25 gm of succinylated silica gelcharacterized by the formula: ##STR34##

200 mg of EDC:HCl was added to this mixture which was thereafteragitated for 3 hours at room temperature. The pH of the solution wasthen adjusted to 4.3 and the mixture allowed to stand overnight. Afterfiltration, the obtained composite was washed thoroughly with distilledwater until the washing water was colorless. Thereafter, the obtainedcomposite was dried under vacuum.

EXAMPLE 8 Immobilization Of Enterobactin To Aminoarylcarbonyl ActivatedSilica Gel

5 gm of aminoarylcarbonyl activated silica gel obtained as in step (c)of example 1, was mixed with 10 ml of 1M sodium nitrite and cooled in anice bath. 1 ml of concentrated hydrochloric acid was added dropwise tothe cooled solution. The mixture was maintained in the ice bath for anadditional 45 min. to allow for complete diazotization. The mixture wasthen filtered in the cold, washed with cold distilled water and, whilestill cold, the gel was added to a solution of about 30 mg ofenterobactin in 10 ml of ethanol including about 2 ml of saturated boraxsolution. The reaction was allowed to proceed for 1 hr in an ice bath.The composite was then recovered by filtration and washed with anethanol water mixture containing 0.1 normal hydrochloric acid andthereafter with ethanol until the filtrate obtained was colorless. Theobtained composite was then dried under vacuum.

The coupling reaction can be represented graphically as below: ##STR35##

EXAMPLE 9 Immobilization Of Cathechol To Polyacrylamide Gel: DiazoCoupling

20 ml of the gel obtained in accordance with Example 2, was suspended in20 ml of 0.1 normal HCl and cooled in an ice bath. While the suspensionwas maintained in contact with the ice bath, 2 ml of 1M sodium nitritewas added dropwise with agitation of the suspension. The resultantmixture was kept in the ice bath for an additional 30 min. andcentrifuged to remove the liquid phase. The gel was then washed twicewith ice-cold distilled water and then placed in contact with 20 ml ofan ice-cold solution of 10% catechol in saturated borax. The reactionwas allowed to proceed overnight at 4° C. The composite was recovered byfiltration and it was washed repeatedly with water containing 0.1 normalHCl. The reaction can be indicated schematically as follows: ##STR36##wherein PA is polyacrylamide carrier or backbone.

EXAMPLE 10 Immobilization Of Catechol To Aminoaryl Carbonyl ActivatedSilica Gel

20 gm of aminoarylcarbonyl activated silica gel obtained in accordancewith example 1 (c) was suspended in 20 ml of ice-cold water. 10 ml of 1Msodium nitrite was added to the suspension while maintaining thesuspension in an ice bath. 10 ml of 2 normal hydrochloric acid(ice-cold) was slowly added dropwise to the suspension. The reaction wasallowed to proceed at between 0° and 4° C. for about 1 hour and it wasthen washed with ice-cold distilled water. The solid was then added to10 ml of 10% cathecol in saturated borax solution (ice-cold) withmixing. 20 ml of water was added to facilitate mixing and the reactionwas allowed to proceed in an ice bath for an additional one hour. Thecomposition was then allowed to settle and the liquid siphoned off. Thecomposition was washed with distilled water containing 0.1 normalhydrochloric acid and ethanol. The composition was repeatedly washeduntil the washing water was colorless. The composition was thenrecovered and dried under a vacuum.

The reactions involved with the catechol can be represented generally asbelow: ##STR37##

EXAMPLE 11 Immobilization Of Catechol To Aldehyde Activated Silica Gel

5 gm of aldehyde activated silica gel (activated as in examle 1 (e)above) was suspended in 10 ml of 10% cathecol in saturated boraxsolution for 1 hour. The mixture was then subjected to vacuumevaporation and then heated while still under vacuum at 70° C. for onehour. The mixture was then cooled to room temperature and water wasadded thereto, the mixture then being heated at 70° C. for an additionalhour. The mixture was then cooled to room temperature and 500 mg ofsodium borohydride was added and the mixture was maintained at 70° C.for a further hour. The resultant reaction mixture was then cooled in anice bath and 5 ml of glacial acetic acid was added dropwise with mixing.The reaction was allowed to proceed for an additional 30 min. and themixture was then filtered. The recovered composition was then washedrepeatedly with distilled water and ethanol alternately and then driedunder vacuum.

The general chemical reactions are believed to be as follows: ##STR38##

EXAMPLE 12 Removal Of Iron From Wine

The inability of wine to spoil after extraction of iron using thecomposition of the present invention is illustrated in table 3 below.This protection from spoilage is retained even when the wine isvigorously aerated (agitated continually in open flasks at 25° C.). Thetreatment of the commercial wine samples consisted of vigorouslyaerating the sample after inoculating with acetobacter xylinum, aerationcontinuing for a period of about 30 days at 25° C.

                  TABLE 3                                                         ______________________________________                                        TREATMENT       OBSERVATIONS                                                  ______________________________________                                        None            By three weeks, wine was foul                                                 smelling (including acetic                                                    acid smell); turbid from bacterial                                            growth                                                        Filter sterilized (no bacteria                                                                No spoilage                                                   added (0.45 μm pore size)                                                  Iron extraction with                                                                          No spoilage                                                   siderophoric composition                                                      of the present invention                                                      Addition of iron ions                                                                         By three weeks, wine was foul                                 to wine subjected to                                                                          smelling (including acetic acid                               iron removal by treat-                                                                        smell); turbid from bacterial                                 ment with the sidero-                                                                         growth                                                        phoric composition of                                                         the present invention                                                         ______________________________________                                    

The following examples (i.e. 13 and 14) illustrate the procedure whichmay be used to recycle siderophoric compositions in accordance with thepresent invention i.e. recycling after removal of bound iron. The reuseof the siderophoric composition of the present invention makes iteconomically attractive.

EXAMPLE 13 Regeneration Of An Active Iron-Free Siderophoric CompositionComprising Enterobactin Fixed To Silica Gel

The siderophoric composition subjected to regeneration can in general berepresented by the following graphic formula: ##STR39## The abovesiderophoric composition loaded with iron, was subjected to treatmentwith an equal volume of 0.1M sodium citrate and 0.1M ascorbic acid, thetreatment lasting for a period of about 12 hours. The treatment wasrepeated twice for a recovery of loaded iron in the range of 95%.

Repeated loading and unloading of the siderophoric composition with Fe³⁺results in retention of up to 95% of the original iron-buildingcapacity.

On average, each gram of iron-loaded siderophoric composition includedabout 212 to 232 micrograms of iron per gram of composition. In theabove procedure, about 80-100 mg. of siderophoric composition weresubjected to regeneration.

EXAMPLE 14 Regeneration Of A Siderophoric Composition Comprising ACatechol Fixed To A Silica Gel

(a) The siderophoric composition can be represented generally by thefollowing graphic formula: ##STR40## The siderophoric composition wassubjected to the same treatment as in Example 13. The iron-loadedsiderophoric composition contained from 110 micrograms to 163 microgramsof Fe³⁺ per gram of the composition. Retention of iron-binding capacityafter regeneration was up to 95%.

(b) The siderophoric composition having the following general structurewas subjected to a reductive regeneration: ##STR41## The iron loadedsiderophoric composition contained from 43 micrograms to 56 microgramsor iron per gram of siderophoric composition. The siderophoriccomposition was regenerated using 5 ml of 0.1M sodium dithionite.Treatment in this way resulted in the recovery of 85 to 93% of theiron-binding capacity of the siderophoric composition.

EXAMPLE 15 A Siderophoric Composition Comprising Ferrioxamine Fixed toSilica Gel

The siderophoric composition was obtained in accordance with theprocedure outlined in Example 7. The siderophoric composition had aniron binding capacity of about 740 micrograms of iron per gram ofsiderophoric composition. 12 gm of the above composition when exposed to5 ml of a 500μM Fe³⁺ solution was able to remove or recover 79.1% of theiron; on being exposed to about 5 ml of a 5μM Fe³⁺ solution about 98.8%of this iron was removed or recovered from the solution by thesiderophoric composition.

EXAMPLE 16 Activation of Biogel P 150 with Ethylenediamine: ##STR42##PA=polyacrylamide back bone:

All operations were carried out in a well ventilated hood. 200 ml.anhydrous ethylene diamine in a 500 ml. 3 necked round bottom flask washeated with a heating mantle and the final temperature was reached andadjusted and maintained at 90° C.±2° C. The glass was also equipped witha condenser with outlet protected by a drying tube, a mechanical stirrerand the third neck was used for addition of materials and temperaturemonitoring. 10 gm of Biogel-P 150 was added through the thermometer neckin one portion and the mixture was stirred and heated at 90° C.±2° C.for a period of 3 to 4 hours. The solid gel swelled to a great volumeand the evolution of ammonia can be ascertained by wetted pH paper atthe drying-tube outlet. At the end of the reaction, the mixture waspoured with mechanical stirring on to a mixture of 400 ml, ice and water(1:1). Any gel adhering to the flask can be washed down by jets ofwater. The gel was filtered while the mixture was still cold. The gelwas promptly washed repeatedly with 0.2M NaCl and 0.001N HCl until thefiltrate gave a negative TNBS test (Trinitrobenzenesulfonic Acid). Thetotal gel volume was about 170 ml. i.e. wet gel.

EXAMPLE 17 Succinylation: Carboxylic Arm Extension: ##STR43##PA=polyacrylamide back-bone.

50 ml. wet gel (Biogel P-150-Ethylenediamine activated) is suspended in50 ml. 0.1N NaOH in a 250 ml. beaker. External cooling in an ice bathand gentle mechanical stirring is also provided. 1.0 gm. succinicanhydride (10 mmole) was added in one portion and the mixture stirred inthe cold for 2 hrs. A further 1 gm. portion of succinic anhydride wasadded with further cooling and stirring for an additional hr. During theaddition of the second portion of succinic anhydride, the mixture pH ismonitored intermittently with a pH meter and additional amounts of 1NNaOH were added to maintain a pH of 3.5 to 4.0. A third portion of 1 g.succinic anhydride was added and the monitoring procedure was same asthe previous addition. The TNBS Test showed that there were still freeamino group on the gel and these were blocked by addition 10 ml. aceticanhydride and stirred for 30 min. The mixture was eventually washedthoroughly with 0.1M NaCl. TNBS Test was negative for the gel.

For the following examples 18 to 21, the formula for the startingcatechol composition is indicated as a matter of convenience ascomprising a single isomer; the composition used was however a mixtureof ##STR44##

EXAMPLE 18 Preparation of a Chloro Substituted Catechol Composition##STR45##

To one batch (prepared from 500 gm silica) of silicat was added 100 mlCommercial sodium hypochlorite (4-6% solution) and the mixture wasstirred and cooled in an ice bath. 50 ml of glacial acetic acid wasadded and the mixture was stirred and evacuated simultaneously. Coolingin the ice bath continued for another 30 minutes. Then 50 mlconcentrated hydrochloric acid was added and the mixture allowed to cometo room temperature gradually. Stirring and evacuation continued for afurther two hours and the mixture was then diluted with a large volumeof water and filtered on a Buchner funnel. The solid was washedthoroughly with water first. Then it was soaked in 5% aqueous ammoniaand washed with water again thoroughly. Then it was soaked in 5%hydrochloric acid and washed with water again. The solid is stored as asuspension in the acidified form for subsequent utilization.

EXAMPLE 19 Preparation of a Bromine Substituted Catechol Composition##STR46##

To one batch (prepared from 500 gm. silica) of silicat was added 10 gm.sodium acetate and the mixture was cooled in an ice bath. This wasfollowed by the addition of 5 ml. of liquid bromine and the mixture wasstirred, evacuated and cooled in an ice bath simultaneously. Thereaction was allowed to proceed in this manner for two hours and theobtained mixture was then washed thoroughly with water to eliminate allunreacted bromine. The mixture was then soaked in 5% ammonium hydroxide,washed with water and then soaked in 5% hydrochloric acid. It wasfinally washed thoroughly with water and stored in water in an acidifiedaqueous environment.

EXAMPLE 20 Preparation of NO Substituted Catechol Composition ##STR47##

One batch (prepared from 500 gm. silica) of silicat was cooledsufficiently in an ice bath and followed by the addition of 7.0 gm.sodium nitrite (NaNO₂). The suspension was stirred, evacuated and cooledin an ice bath simultaneously. The reaction was allowed to proceed for30 minutes. This was followed by the addition of 10 ml. concentratedhydrochloric acid and a perceptible evolution of nitrogen oxide wasobserved. The reaction was processed as before for another two hours.The mixture was then washed thoroughly with water, soaked in 5% ammoniumhydroxide which brings about a deepening of the color of the solid to aredder shade. The filtrate and aqueous washings were reddish orange andwashing with water continued until the washings were colorless. Thesolid was then soaked in 5% hydrochloric acid which changes the colorfrom reddish to a more orange shade and the suspension was washedthoroughly with water and stored in this acidified form.

EXAMPLE 21 Preparation of NO₂ Substituted Catechol Composition ##STR48##

One batch (prepared from 500 gm. silica) of silicat was cooled in an icebath thoroughly. To this cooled suspension was added continuously 250ml. commercial concentrated nitric acid and the mixture was stirred,evacuated and cooled in ice bath for 30 min. Then 250 ml. concentratedsulfuric acid was added very cautiously with careful stirring. Themixture was warmed up considerably and it was cooled in the ice bath.The reaction was allowed to proceed for another two hours. The mixturewas then diluted with a large amount of water and then filtered, washedwith water, followed by soaking with 5% ammonium hydroxide, giving amixture reddening in shade and orange filtrates and washings. Thoroughwashing with water was continued until the washing was colorless. It wasthen soaked in 5% hydrochloric acid and which brought about a lighteningof color from reddish to orange and washed with water again thoroughly.It was stored in this acidified form for subsequent utilization.

EXAMPLE 22 Removal of UO₂ ²⁺ from Solution Using a CompositionConsisting of Desferrioxamine Fixed to a Silica Gel

A bed consisting of 4 gms of a composition consisting of desferrioxaminecoupled to a silica gel with glutaraldehyde (see example no. 1(e) wasplaced into a column. The composition was then treated with 100 ml of anaqueous 0.5M sodium dithionite solution to remove any bound metal e.g.iron. The composition was then washed with 100 ml of an aqueous 0.01Msodium acetate solution of pH 5.9 at 20° C. The composition was thencontacted at 20° C. with a total of 80 ml of an aqueous solution (pH6.9) containing Uranyl acetate at a concentration of 600μM the solutionbeing brought into contact with the composition at the rate of 2.3ml/min. No uranyl ion emerged from the column until a total of 62 ml ofthe solution had been applied. The composition took up a total of 34.6μmole of UO₂ ²⁺ or 8.64μ mole of UO₂ ²⁺ (2.0 mg of UO₂ ²⁺) per gram ofcomposition. The loaded composition was thereafter treated with anaqueous 6 mM Fe³⁺ solution (pH 6.9). The composition failed to develop ared colour indicative of the binding of Fe³⁺ thereto; i.e. compositionhas higher affinity for UO₂ ²⁺ than for Fe³⁺.

EXAMPLE 23 Removal of UO₂ ²⁺ from a Citrate Buffered Solution Using aComposition Consisting of Catechol Fixed to a Silica Gel

110 ml. of a buffered uranyl acetate solution (Uranyl acetate 600μM;sodium acetate 60 mM) of pH 6.9 was contacted with 4 gms (dry wgt) of acatechol-glutaraldehyde-silica composition (see example no. 11). Thecomposition was >99.9% efficient in removing Uranyl ion from solutionfor the first 50 ml of solution contacted with the composition; i.e. thecomposition took up to 7.3μ mol UO₂ ²⁺ /gm of composition. Theefficiency of the composition thereafter progressively decreased. Theoverall binding capacity of the composition was found to be about 14μM(3.3 mg) UO₂ ²⁺ /gm of composition. Overall the concentration of theUranyl ion in the solution was lowered to less than 6μM from 600μM.

EXAMPLE 24 Removal of UO₂ ²⁺ in Trace Amounts from Distilled Water Usinga Composition Consisting of Catechol Fixed to a Silica Gel.

42 l. of water containing 285 nM UO₂ ²⁺ acetate (˜pH 6.9) was contactedwith 30 gm (dry weight) of a catechol-glutaraldehyde-silica composition(see ex. no. 11) placed in a column, at a rate of 4.5 ml/min; thecomposition was preconditioned by contact with 100 ml of aqueous 0.1Msodium acetate solution pH 6.9. The loaded composition was then washedwith 50 ml aqueous 2N HCl to wash out the UO₂ ²⁺. It was determined thatthe amount of UO₂ ²⁺ removed from the water was such that the 285 nMsolution was lowered to <0.2 nM; i.e. an efficiency of >99.9%.

EXAMPLE 25 Removal of UO₂ ²⁺ from Artificial Sea Water Using aComposition Consisting of Catechol Fixed to a Silica Gel

30 gm (dry weight) of a catechol-glutaraldehyde-silica composition (seeex. No. 11) was placed in a column and contacted with 100 ml of anaqueous 0.1M sodium acetate solution at pH 6.9. 900 ml of 13μM UO₂ ²⁺acetate in artificial sea water (forty fathoms) adjusted to pH 6.6 wasthereafter contacted with the composition. The composition was thenwashed with 200 ml of distilled water. The washed composition was thencontacted with 100 ml aqueous 2N HCl to wash out the UO₂ ²⁺. Of the 12μMole of UO₂ ²⁺ in the 900 ml of sea water 11.52 were recovered from thecomposition that could be eluted with the 2N HCl; (i.e. recoveryefficiency of ≈96%).

EXAMPLE 26 Elution of UO₂ ²⁺ from a Composition Consisting of CatecholFixed to a Silica Gel to Obtain the UO₂ ²⁺ in a Concentrated Solution

4 gm (dry wgt) of a catechol-glutaraldehyde-silica composition (see exno. 11) was loaded with UO₂ ²⁺ by contact with 50 ml of an aqueoussolution (600μM UO₂ acetate in 0.1M Na acetate) at pH 6.9. The loadedcomposition was washed with 100 ml of distilled water and then washedwith 50 ml aqueous 2N HCl to wash out the UO₂ ²⁺. 99.94% of the UO₂ ²⁺bound to the composition was removed.

EXAMPLE 27 Removal of Th⁴⁺ from a Buffered Solution Using a CompositionConsisting of Catechol Fixed to a Silica Gel

4 gm (dry weight) of a catechol-glutaraldehyde-silica composition (seeex. no. 11) was placed in a column and treated with 100 ml of an aqueous0.1M Na acetate solution pH 6.9. The composition was then contacted with100 ml of an aqueous solution (600μM Th (NO₃)₄ in 6 mM Na citrate) pH6.9 at a rate of 3 ml/min. The results were nearly identical to those inex. 22.

EXAMPLE 28 Separation of UO₂ ²⁺ and Th⁴⁺ Bound to a CompositionConsisting of Catechol Fixed to a Silica Gel

30 gms (dry wt) of a catechol-glutaraldehyde-silica composition (seeexample no. 11) was loaded with UO₂ ²⁺ and Th⁴⁺ by contacting it with100 ml of an aqueous solution containing

    ______________________________________                                        Th(NO.sub.3).sub.4    3     mM                                                UO.sub.2 (acetic).sub.2                                                                             3     mM                                                Na.sub.3 (Citrate)    60    mM                                                pH                    6.9                                                     ______________________________________                                    

The loaded composition was washed with distilled water and thereafterplaced in a column. The composition was then washed with 200 ml ofaqueous 60 mM Na Citrate pH 6.9. The composition was then eluted with acontinuous pH gradient starting from pH 6.9 and finishing at pH 0.8. Thegradient was prepared by starting with an aqueous 0.1M sodium citratesolution of pH 6.9, thereafter mixing said citrate solution with anaqueous 0.3N HCl (the citrate in lower and lower amounts the HCl inhigher and higher amounts) until finally finishing with said aqueous0.3N HCl solution pH 0.8. Over all a total of 150 ml of each solutionwas used. The Th⁴⁺ was selectively eluted at a pH of about 4.45. The UO₂²⁺ was eluted at the lower end of the pH gradient.

We claim:
 1. A method for inhibiting microbial growth in a liquidnutrient medium containing Fe³⁺ by lowering the Fe³⁺ content thereof toless than 0.1μM characterized in that said medium is contacted with aninsoluble siderophoric composition and thereafter said siderophoriccomposition loaded with Fe³⁺ is separated from said medium, saidinsoluble siderophoric composition comprising:(1) one or more organicsiderophoric compounds covalently fixed to the surface of (2) a suitableinsoluble carrier, said organic siderophoric compounds possessing one ormore coordinating sites capable of chelating Fe³⁺.
 2. A method asdefined in claim 1, wherein said coordinating sites are provided by oneor more members of the class consisting of(a) a N-substitutedhydroxamate group of formula ##STR49## (b) a phenolate group of formula##STR50## X being an atom of O or N--, and (c) a catecholate group offormula ##STR51## X' being an atom of O or N-and m being
 1. 3. A methodfor inhibiting microbial growth in a liquid nutrient medium containingFe³⁺ by lowering the Fe³⁺ content thereof to less then 0.1μMcharacterized in that said medium is contacted with an insolublesiderophoric composition and thereafter said siderophoric compositionloaded with Fe³⁺ is separated from said medium, said insolublesiderophoric composition comprising(1) one or more organic siderophoriccompounds covalently fixed to the surface of (2) a suitable insolublecarrier, said organic siderophoric compounds possessing one or morecoordinating sites capable of chelating Fe³⁺, said organic siderophoriccompounds being selected from the class consisting of microbialsiderophores.
 4. A method as defined in claim 3, wherein said microbialsiderophores have a molecular weight of less then 2500 daltons.
 5. Amethod as defined in claim 4, wherein said coordinating sites areprovided by one or more members of the class consisting of(a) aN-substituted hydroxamate group of formula ##STR52## (b) a phenolategroup of formula ##STR53## X being an atom of O or N--, and (c) acatecholate group of formula ##STR54## X' being an atom of O or N-- andm being
 1. 6. A method as defined in claim 3, wherein said microbialsiderophores have a molecular weight in the range of 500 to 2500daltons.
 7. A method as defined in claim 6, wherein said coordinatingsites are provided by one or more N-substituted hydroxamate groups offormula ##STR55##
 8. A method as defined in claim 6, wherein saidcoordinating sites are provided by one or more catecholate groups offormula ##STR56##
 9. A method as defined in claim 6, wherein saidcoordinating sites are provided by one or more members of the classconsisting of(a) a N-substituted hydroxamate group of formula ##STR57##(b) a phenolate group of formula ##STR58## X being an atom of O or N--,and (c) a catecholate group of formula ##STR59## X' being an atom of Oor N-- and m being
 1. 10. A method as defined in claim 3, wherein saidcoordinating sites are provided by one or more members of the classconsisting of(a) a N-substituted hydroxamate group of formula ##STR60##(b) a phenolate group of formula ##STR61## X being an atom of O or N--,and (c) a catecholate group of formula ##STR62## X' being an atom of Oor N-- and m being
 1. 11. A method as defined in claim 3, wherein saidorganic siderophoric compounds are selected from the class consisting ofdesferrioxamines.
 12. A method as defined in claim 3, wherein theorganic siderophoric compound covalently fixed to the carrier isenterobactin.
 13. A method for inhibiting microbial growth in a liquidnutrient medium containing Fe³⁺ by lowering the Fe³⁺ content thereof toless then 0.1μM characterized in that said medium is contacted with aninsoluble siderophoric composition and thereafter said siderophoriccomposition loaded with Fe³⁺ is separated from said medium, saidinsoluble siderophoric composition comprising(1) one or more catecholcompounds covalently fixed to the surface of (2) a suitable insolublecarrier, said catechol compounds being covalently fixed to the surfaceof said carrier at the benzene ring thereof, said catechol compoundsbeing selected from the group consisting of catechol substituted on thebenzene ring by one or two electrophilic substituents.
 14. A method asdefined in claim 13, wherein the catechol compound covalently fixed tothe carrier is unsubstituted catechol.
 15. A method as defined in claim13, wherein said catechol compounds are selected from the groupconsisting of catechol substituted on the benzene ring by one or twosubstituents selected from the class consisting of halogen atoms, NO andNO₂.
 16. A method as defined in claim 13, wherein said catecholcompounds are selected from the group consisting of catechol substitutedon the benzene ring by one or two halogen atoms.
 17. A method as definedin claim 16, wherein said halogen atoms are selected from chlorine andbromine.
 18. A method as defined in claim 13, wherein said catecholcompound is selected from the group consisting of catecholmonosubstituted on the benzene ring by a substituent selected from NOand NO₂.
 19. A method for removing Fe³⁺, Th⁴⁺, UO₂ ²⁺ or mixturesthereof from solution characterized in that the solution is contactedwith an insoluble composition comprising a member selected from theclass consisting of(A) an insoluble composition comprising(1) one ormore organic chelating compounds, covalently fixed to the surface of (2)a suitable insoluble carrier, said organic chelating compoundspossessing one or more coordinating sites, said organic chelatingcompounds being selected from the class consisting of microbialsiderophores and (B) an insoluble composition comprising(1) one or morecatechol compounds covalently fixed to the surface of (2) a suitableinsoluble carrier, said catechol compounds being covalently fixed to thesurface of said carrier at the benzene ring thereof, said catecholcompounds being selected from the group consisting of unsubstitutedcatechol and catechol substituted on the benzene ring by one or twoelectrophilic substituents.
 20. A method for removing Fe³⁺, Th⁴⁺, UO₂ ²⁺or mixtures thereof from solution characterized in that the solution iscontacted with an insoluble composition comprising(1) one or moreorganic chelating compounds, covalently fixed to the surface of (2) asuitable insoluble carrier, said organic chelating compounds possessingone or more coordinating sites, said organic chelating compounds beingselected from the class consisting of microbial siderophores.
 21. Amethod as defined in claim 20, wherein said microbial siderophores havea molecular weight of less than 2500 daltons.
 22. A method as defined inclaim 21, wherein said coordinating sites are provided by one or moremembers of the class consisting of(a) a N-substituted hydroxamate groupof formula ##STR63## (b) a phenolate group of formula ##STR64## X beingan atom of O or N--, and (c) a catecholate group of formula ##STR65## X'being an atom of O or N-- and m being
 1. 23. A method as defined inclaim 20, wherein said microbial siderophores have a molecular weight inthe range of 500 to 2500 daltons.
 24. A method as defined in claim 23,wherein said coordinating sites are provided by one or moreN-substituted hydroxamate groups of formula ##STR66##
 25. A method asdefined in claim 23, wherein said coordinating sites are provided by oneor more catecholate groups of formula ##STR67##
 26. A method as definedin claim 23, wherein said coordinating sites are provided by one or moremembers of the class consisting of(a) a N-substituted hydroxamate groupof formula ##STR68## (b) a phenolate group of formula ##STR69## X beingan atom of O or N--, and (c) a catecholate group of formula ##STR70## X'being an atom of O or N-- and m being
 1. 27. A method as defined inclaim 20, wherein said coordinating sites are provided by one or moremembers of the class consisting of(a) a N-substituted hydroxamate groupof formula ##STR71## (b) a phenolate group of formula ##STR72## X beingan atom of O or N--, and (c) a catecholate group of formula ##STR73## X'being an atom of O or N-- and m being
 1. 28. A method as defined inclaim 20, wherein said organic chelating compounds are selected from theclass consisting of desferrioxamines.
 29. A method as defined in claim20, wherein the organic chelating compound covalently fixed to thecarrier is enterobactin.
 30. A method for removing Fe³⁺, Th⁴⁺, UO₂ ²⁺ ormixtures thereof from solution characterized in that the solution iscontacted with an insoluble composition comprising:(1) one or morecatechol compounds covalently fixed to the surface of (2) a suitableinsoluble carrier, said catechol compounds being covalently fixed to thesurface of said carrier at the benzene ring thereof, said catecholcompounds being selected from the group consisting of unsubstitutedcatechol and catechol substituted on the benzene ring by one or twoelectrophilic substituents.
 31. A method as defined in claim 30, whereinthe catechol compound fixed to the carrier is unsubstituted catechol.32. A method as defined in claim 30, wherein said catechol compounds areselected from the group consisting of catechol substituted on thebenzene ring by one or two substituents selected from the classconsisting of halogen atoms, NO and NO₂.
 33. A method as defined inclaim 30, wherein said catechol compounds are selected from the groupconsisting of catechol substituted on the benzene ring by one or twohalogen atoms.
 34. A method as defined in claim 33, wherein said halogenatoms are selected from chlorine and bromine.
 35. A method as defined inclaim 30, wherein said catechol compound is selected from the groupconsisting of catechol monosubstituted on the benzene ring by asubstituent selected from NO and NO₂.
 36. A method as defined in claim30, wherein said catechol compounds convalently fixed to the carrier areselected from the group consisting of catechol substituted on thebenzene ring by one or two electrophilic substituents.
 37. A method asdefined in claim 36, wherein said electrophilic substituents areselected from the group consisting of halogen atoms, NO, NO₂, COOH and##STR74##
 38. A method for removing Th⁴⁺ from solution comprising thestep of contacting a solution which contains Th⁴⁺ with an insolublecomposition comprising catechol convalently fixed to the surface of asuitable insoluble carrier, said catechol being fixed at the benzenering thereof to the surface of said carrier.